Advanced cell-transducing transport domain-target protein-transport domain fusion protein and uses thereof

ABSTRACT

The present invention provides a fusion protein which delivers a functional protein or peptide into a cell at enhanced efficiency. The fusion protein of the present invention is a transduction domain-target protein-transduction domain fusion protein, wherein the transduction domain, which comprises 6-12 amino acid residues whose more than ¾ consist of arginine or lysine residues, is covalently bonded to each of the amino- and carboxyl-terminal ends of the target protein. Green fluorescence protein and Cu/Zn-superoxide dismutate (SOD) are used as the target protein.

TECHNICAL FIELD

The present invention relates to a fusion protein of delivering afunctional protein or peptide into a cell at increased efficiency, anduses thereof.

BACKGROUND ART

Recently, as the fact that various diseases are caused by the abnormalactivity of cell proteins is known, the development of drugs capable ofadjusting the activity of such proteins to treat fatal human diseasesbecomes the object of interest in the whole world.

In a description of the present invention, superoxide dismutase relatedto reactive oxygen species is mentioned as one example of the cellproteins. However, it is to be understood that the scope of the targetproteins in the present invention is not limited only to this protein.

Reactive oxygen species are inevitably produced as byproducts ofintracellular metabolism in all living beings where energy is obtainedusing oxygen. Such reactive oxygen species cause damage to biopolymers,such as intracellular protein, nucleic acid and fat, and have a deepconnection with the progression of various diseases of the human body.Particularly, they are involved in carcinogenesis processes, apoplexy,arthritis, radiation damage and inflammatory reaction, and act as animportant factor of promoting aging even in a normal aging process[Floyd, R. A., FASEB J. 4, 2587-2597 (1990); Anderson, W. F., Human genetherapy, Nature 392, 25-30 (1998); and Halliwell B. and Gutteridge J. M.C., Free radicals in biology and medicine, Oxford University Press,Oxford (1999)].

Diseases related to Cu/Zn-superoxide dismutase are summarized in Table 1below.

TABLE 1 Categories Concrete examples Inflammatory/ Glomerulonephritis,vasculitis, autoimmune immune damage diseases and the like IschemiaStroke, myocardial infarction, arrhythmia, angina pectoris and the likeDrug and toxic — substance-inducing reaction Iron overload Idiopathichemochromotosis and the like (tissue and serum) Radiation-related damageNuclear bombing, radiation therapy and the like Aging Progeria, anddisease-related aging Red blood cells Sickle-cell anemia, malaria andthe like Bronchi A result of smoking, emphysema and the like Heart andCardiomyopathy cardiovascular systems Kidneys Autoimmune nephroticsyndrome Stomach and intestines Betelnut-related oral cancer Abnormalconditions Hypoxia, Alzheimer's disease, Parkinson's of brains/nervousdisease and the like system/nervous muscles Eye Cateract and the likeSkin Diseases caused by UV irradiation, and the like *Quoted from “Freeradicals in biology and medicine”, Oxford University Press, Oxford, pp618-619.

Known reactive oxygen species include ₁O₂, OH, O₂, H₂O₂ and the like.They are produced by various enzymatic reactions, and play an importantrole in the biosynthesis and immune function of various physiologicallyactive substances, and drug metabolism. However, if they areover-produced by external radiation, UV, environmental pollution andvarious stresses, they can rather cause damage to the living body. Forthis reason, the living body has enzymes, such as SOD, catalase andperoxidase, for their defensive function, and if its aging is started,the balance of the skin will be upset and the ability of this enzyme toprotect the skin from various reactive oxygen species will be reduced.

Thus, a need to protect the skin from such reactive oxygen species isbeing increased, and SOD, lactoferrin, and antioxidants, etc., are usedor developed as raw materials of cosmetics for the removal of thereactive oxygen species. However, SOD was not advantageously employed ina cosmetic composition in spite of its ability to remove the reactiveoxygen species, because it has the nature of enzymatic protein and thusproblems in that it is difficultly soluble in lipid, has insufficientstability, and is impermeable to the skin's keratin layer due to itsmolecular weight of more than 30,000 Daltons.

Cu/Zn-superoxide dismutase is an important intracellular defensiveenzyme of preventing the cellular damage caused by free radical toxicityand oxygen-radical damage [Fridovich, I., Annu. Rev. Biochem., 64,97-112 (1995)]. Since all polymers in the living body are always exposedto this harmful action of the oxygen radical, an interest to use theCu/Zn-superoxide dismutase for the treatment of various diseases isbeing increased.

Recently, there are many attempted methods for clinically applying theCu/Zn-superoxide dismutase. Methods for delivering the Cu/Zn-superoxidedismutase into the living body, which have been developed till now, canbe broadly classified into the following three categories. First, thereis a method of conjugating polyethylene glycol, ficoll, lecithin,albumin and the like to the Cu/Zn-superoxide dismutase [Del Zoppo, G. J.et al., Drugs 54, 9-38, (1997); and Muzykantov, V. R. et al., Proc.Natl. Acad. Sci. USA 93, 5213-5218, (1996)]. Second, there is a methodof encapsulating the Cu/Zn superoxide dismutase with liposome[Perdereau, B. et al., Bull. Cancer 81, 659-669 (1994)]. Third, there isa genetic therapy where the Cu/Zn superoxide dismutase gene istransduced into cells to induce the overexpression of the enzyme in thecells [Okumura, K. et al., Pharm. Res. 14, 1223-1227; (1997); Lehmann,T. G. et al., Transplantation 69, 1051-1057 (2000); and Liu, R. et al.,Hum. Gene Ther. 8, 585-595 (1997)].

Among such methods, the genetic therapy is most attracted, and manystudies to use the Cu/Zn-superoxide dismutase gene for the treatment ofdiseases have been conducted. However, the genetic therapy has variousproblems in that a method of delivering the gene into a cell is noteasy, the percent expression of the gene in a target cell is low, and itis very difficult to artificially adjust the amount of expression of theprotein in the target cell [Verma, I. M. et al., Nature 389, 239-242(1997)].

As another method of delivering a therapeutic drug or protein into acell, a method of directly delivering a target protein through a cellmembrane to a cell can be contemplated. However, the therapeutic drug orprotein is very difficult to pass through the cell membrane due to itssize or various biochemical properties. It is generally known thatsubstances with a molecular weight above 600 are almost impossible topass through the cell membrane.

It was recently found that a Tat (transactivator of transcription)protein as a kind of human immunodeficiency virus type-1 proteins isefficiently passed through the cell membrane such that it is easilydelivered into a cytoplasm. This function appears due to the property ofa protein transduction domain as the middle domain of the Tat protein,and its precise mechanism is yet unknown [Frankel, A. D. and Pabo, C.O., Cell 55, 1189-1193 (1988); Green, M. and Loewenstein, P. M., Cell55, 1179-1188 (1988); Ma, M. and Nath, A., J. Virol. 71, 2495-2499(1997); and Vives, E., Brodin, P. and Lebleu, B., J. Biol. Chem. 272,16010-16017 (1997)]. However, it seems that a certain receptor orcarrier is not involved in the passage of the Tat protein through thecell membrane, and this passage of the Tat protein is caused by thedirect interaction between the protein transduction domain of the Tatprotein and the lipid double layer of the cell membrane [Vives, E. etal., J. Biol. Chem. 272, 16010-16017 (1997); and Derossi, D. et al., J.Biol. Chem. 271, 18188-18193 (1996)].

Recent studies showed that when heteroproteins, such as ovalbumin,β-galactosidase, and horseradish peroxidase, was administered in a formfused with an HIV-1 Tat protein, they were delivered directly into eachtissue of the living body and a cultured cell [Fawell, S. et al., Proc.Natl. Acad. Sci. USA 91, 664-668 (1994); Schwartze, S. R. et al.,Science. 285, 1569-1572 (1999); and Watson, K. and Edward, R. J.,Biochem. Pharmacol., 58, 1521-1528 (1999)]. This test result suggeststhat the Tat protein has the ability to deliver itself and also othermacromolecules into the cell.

However, it is not that substantially all proteins are delivered by theTat protein. Furthermore, whether all the proteins delivered by the Tatprotein into the cell show biological activity is not yet certainlyestablished.

Moreover, the present inventors conducted an intracellular transductiontest for the following fusion proteins: a fusion protein where a HIV Tatprotein transduction domain (residues 49-57) is covalently bonded to theamino-terminal end, a fusion protein where an oligolysine transductiondomain having 6-12 lysines are covalently bonded to the amino-terminalend; and a fusion protein where a basic transduction domain from which 2or 3 residues of HIV Tat residues 48-57 had been deleted, are covalentlybound to the amino-terminal end. The test results showed that the fusionproteins had the ability to be smoothly transduced into the cell (Koreanpatent laid-open publication Nos. 2002-10446 and 2002-67108).

DISCLOSURE OF INVENTION

Therefore, an object of the present invention is to deliver or express atarget protein in a cell at high efficiency without encountering theproblems of the prior art, such that the target protein has activity inthe cell.

Another object of the present invention is to deliver or express asuperoxide dismutase in a cell at high efficiency without encounteringthe problems of the prior art, such that the superoxide dismutase hasactivity in the cell.

Still another object of the present invention is to provide cosmeticswhich comprises a transduction domain-superoxide dismutase fusionprotein, so that they are delivered into the epidermal, dermal andsubcutaneous fat layers of the skin by virtue of the fusion protein andthus have the excellent ability to remove reactive oxygen species.

To achieve the above objects, the present inventors fused a transductiondomain to a human Cu/Zn-superoxide dismutase, and in a test using a HeLacell, we have examined if the fusion protein is effectively transducedinto a cell. Furthermore, the present inventors developed a method ofmass-producing and purifying a transduction domain-superoxidedismutase-transduction domain fusion protein.

In order to more efficiently deliver a functional protein or peptideinto a cell and to make the functional protein shows increased activityin the cell, the present invention provides a transductiondomain-functional protein fusion protein, and pharmaceutical andcosmetic compositions using this fusion protein.

The transduction domain-target protein-transduction domain fusionprotein of the present invention can be produced according to a generalchemical bonding method in addition to a genetic recombinant method.

The present invention provides a transduction domain-targetprotein-transduction domain fusion protein having the ability to betransduced into a cell, wherein the transduction domain, which comprises6-12 amino acid residues whose more than ¾ consist of arginine or lysineresidues, is covalently bonded to each of the amino- andcarboxyl-terminal ends of the target protein. The transduction domainslocated at both terminal ends of the target protein do not need toconsist of the same residue.

In the transduction domain-target protein-transduction domain fusionprotein of the present invention, the transduction domain preferablyconsists of 9 amino acid residues.

In the present invention, the transduction domain is preferably one ormore selected from HIV tat residues 49-57, oligolysine, oligoarginine,and oligo(lysine/arginine).

Moreover, the target protein is preferably selected from the groupconsisting of a therapeutic molecule, a preventive molecule, and adiagnostic molecule.

Furthermore, the target protein is preferably a Cu/Zn-superoxidedismutase or functional equivalents thereof.

In another embodiment, the present invention provides a cosmeticcomposition which contains, as an active ingredient, the transductiondomain-target protein-transduction domain fusion protein having theability to be transduced into a cell.

In the cosmetic composition of the present invention, the target proteinis preferably a Cu/Zn-superoxide dismutase or functional equivalentsthereof.

Moreover, the cosmetic composition of the present invention ispreferably in the form of toilet water, gel, water-soluble liquid,oil-in-water (O/W), or water-in-oil (W/O).

In yet another embodiment, the present invention provides apharmaceutical composition which contains the transduction domain-targetprotein-transduction domain fusion protein as an active ingredient, anda pharmaceutically acceptable carrier.

In the pharmaceutical composition of the present invention, the targetprotein is preferably a Cu/Zn-superoxide dismutase or functionalequivalents thereof.

The pharmaceutical composition containing the transduction domain-targetprotein-transduction domain fusion protein as an active ingredient canbe formulated into an oral or injection form by a conventional methodtogether with a pharmaceutically acceptable carrier. Examples of theoral composition include tablets and gelatin capsules, etc. In additionto the active ingredient, the oral composition may, if necessary,contain a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine), and a lubricant (e.g., silica, talc, stearicacid, and magnesium and calcium salts thereof, and/or polyethyleneglycol). The tablets preferably contain a binder (e.g., magnesiumaluminum silicate, starch paste, gelatin, methyl cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone), and if necessary,a disintegrant (e.g., starch, agar, alginic acid or a sodium saltthereof) or a boiling mixture and/or an absorbing agent, a coloringagent, a flavoring agent and a sweetening agent. The injectioncomposition is preferably in the form of isotonic aqueous solution orsuspension, and it is sterilized and if necessary, contains aids(preservatives, stabilizers, wetting agents, emulsifier, accelerators,salts for adjusting osmotic pressure, and/or buffers). In addition, theymay also contain therapeutically useful substances.

The pharmaceutical formulation thus produced can be administered orallyor by parenteral routes, including intravenous, subcutaneous,intra-abdominal or topical routes, at a dosage of 0.0001-2 mg/kg one toseveral times a day. The dosage for a certain patient can vary dependingon the patient's body weight, age, sex and health, the period of timefor administration, percent excretion, and the severity of diseases,etc.

Furthermore, the composition of the present invention can be formulatedinto cream, ointment, gel, lotion, and toilet water, etc., before use,and a form into which the composition is formulated before use can beeasily determined by a person skilled in the art.

Moreover, each of the lotion, gel, essence, cream, toilet water and thelike, which contain the transduction domain-target protein-transductiondomain fusion protein of the present invention as an active ingredient,can be easily produced in any form according to a conventional method,and conveniently added to basic cosmetics before use.

For example, in producing cream, the fusion protein of the presentinvention is added to a general oil-in-water (O/W) or water-in-oil (W/O)cream base, to which perfumes, chelating agents, pigments, antioxidants,preservatives and the like are added in combination with synthetic ornatural materials, such as proteins, minerals and vitamins, for thepurpose of improving the physical properties of the cream.

As used herein, the term “target protein” means a therapeutic,preventive or diagnostic molecule, which forms a covalent bond with theHIV-1 Tat transduction domain and shows activity when delivered into acell. In fact, this term is not limited only to a pure protein, butintended to include peptide, polypeptide, sugar protein bound tosaccarides, peptidoglycan.

As used herein, the term “transduction domain” means a domain whichforms a covalent bond with peptide, protein, oligopeptide, sugar proteinbound to saccharides, peptidoglycan or polypeptide, and allows theorganic compounds to be transduced into a cell without the need of aseparate receptor or carrier, or energy. This transduction domainconsists of 6-12 amino acid residues, more than ¾ of which consist ofarginine or lysine residues. Typical examples of this transductiondomain include HIV-1 Tat (amino acids 48-57), oligolysine,oligoarginine, oligo(lysine/arginine) and the like. A more preferredexample of the inventive transduction domain is one consisting of 9amino acid residues whose more than ¾ consist of arginine or lysineresidues.

In the specification and claims, the term “delivering” protein,oligopeptide, sugar protein, oligopeptide, or polypeptide, etc, into acell, was used exchangeably with the expressions “introducing”,“infiltrating”, “transporting”, “transducing” and “passing”.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of Tat-GFP, GFP-Tat, Tat-GFP-Tat, and GFPexpression vectors. Such vectors were produced by fusing a synthesizedTat transduction domain to the amino-terminal end or carboxyl-terminalend or both terminal ends of GFP. GFP expression vector pGFP wasproduced by inserting the coding sequence of GFP into pET 15b.

FIG. 2 shows a transduction domain-GFP fusion protein which waspurified, isolated by 12% SDS-PAGE, and then stained with Coomassieblue.

Lane M: SDS Marker; lane GFP: GFP; Lane TG: Tat-GFP; lane GT: GFP-Tat;and Lane TGT: Tat-GFP-Tat.

FIG. 3 a shows the results of Western blot analysis to examine theintracellular transduction of denatured Tat-GFP, GFP-Tat, andTat-GFP-Tat fusion proteins. For the Western blot analysis, 1M of thefussion proteins were added to HeLa cells and then the cells werecultured for 1 hour.

FIG. 3 b diagrammatically shows the intensity of line according toWestern blot results for proteins introduced into cells.

FIG. 4 shows the images of Tat-GFP, GFP-Tat, and Tat-GFP-Tat fusionproteins taken with a confocal fluorescent microscope, after theproteins were introduced into cells. Each section is as follows:

control: untreated cell; GFP: 1M control GFP; TG: 1M Tat-GFP 1M; GT: 1MGFP-Tat; and TGT: 1M Tat-GFP-Tat.

FIG. 5 shows the results of FACS analysis to examine the efficiency ofintracellular transduction of GFP fusion proteins. For the FACSanalysis, each of the fusion proteins was added to cells at a 2Mconcentration and then the cells were cultured for 30 minutes.

TG: Tat-GFP; GT: GFP-Tat; and TGT: Tat-GFP-Tat.

FIG. 6 shows the results of FACS analysis for cells, which were addedwith Tat-GFP-Tat fusion protein of various concentrations and culturedfor 30 minutes. Control GFP was added to cells at the maximumconcentration of 2M.

FIG. 7 a shows the results of Western blot analysis to examine theefficiency of intracellular transduction of Tat-GFP-Tat fusion proteinswhich were purified in native and denatured states. For the Western blotanalysis, each of the fusion proteins was added to cells at a 1Mconcentration and then the cells were cultured for 1 hour.

Lane con: an untreated cell; lane GFP: control GFP; lane “native”:Tat-GFP-Tat purified in a native state; and lane “denatured”:Tat-GFP-Tat purified in a denatured state.

FIG. 7 b is a graphic diagram showing the intensity of lines accordingthe results of FIG. 7 a.

FIG. 8 shows the results of FACS analysis to examine the efficiency ofintracellular transduction of Tat-GFP-Tat fusion proteins purified innative and denatured states. For the FACS analysis, the fusion proteinswere added to cells at various concentrations and then the cells werecultured for 1 hour.

FIG. 8 a shows the results of analysis for a Tat-GFP-Tat fusion protein,which was purified in a denatured state and added to cells atconcentrations of 0.1M, 0.2M, 0.5M, and 1M.

FIG. 8 b shows the results of analysis on a Tat-GFP-Tat fusion protein,which was purified in a native state and added to cells atconcentrations of 0.1M, 0.2M, 0.5M, and 1M.

FIG. 9 is a schematic diagram of Tat-SOD, SOD-Tat, Tat-SOD-Tat, and SODexpression vectors. The vectors were produced by fusing a synthesizedTat protein to the amino- or carboxyl- terminal end or both terminalends of SOD. pSOD as an SOD expression vector was produced by insertingthe coding sequence of SOD into a pET-15b vector.

FIG. 10 shows transduction domain-GFP fusion proteins which werepurified, isolated by 12% SDS-PAGE, and then stained with Coomassieblue.

Lane M: SDS marker; lane SOD: SOD; Lane TS: Tat-SOD; lane ST: SOD-Tat;and Lane TST: Tat-SOD-Tat.

FIG. 11 a shows the results of Western blot analysis for intracellulartransduction of Tat-SOD, SOD-Tat, Tat-SOD-Tat fusion proteins, whichwere purified in a denatured state, and then transduced into HeLa cellsat a 2M concentration for 1 hour.

FIG. 11 b is a graphic diagram showing a change in activity of an SODenzyme, which was transduced into HeLa cells at 2M for 1 hour.

FIG. 12 is a graphic diagram showing the results of fluorescentmeasurement to examine the efficiency of intracellular transduction of aTat-GFP-Tat fusion protein and an arginine 9-GFP-arginine 9 (R9-GFP-R9)fusion protein.

FIG. 13 is a fluorescent microscopic photograph showing the comparisonbetween the efficiency of intracellular transduction of a GFP fusionprotein, a Tat-GFP fusion protein, a GFP-Tat fusion protein, an arginine9-GFP fusion protein, and a lysine-GFP-lysine 9 (K9-GFP-K9) fusionprotein. Each of the fusion proteins was added to HeLa cells at 200 nMand then the cells were cultured for one hour.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will hereinafter be described in further detail byexamples. It will however be obvious to a person skilled in the art thatthat the present invention is not limited to or by the examples.

EXAMPLE 1 Production of Transduction Domain Fusion Protein ExpressionVector

To develop a technology of delivering a functional protein or peptideinto a cell, fusion protein expression vectors capable of delivering thetarget protein into the cell were produced.

In this example, to facilitate the analysis of the ability of atransduction domain to deliver a target protein into a cell, a greenfluorescence protein (referred to as “GFP” in the specification) wasselected as a target protein. A DNA fragment corresponding to the basesequence of GFP was subjected to polymerase chain reaction (PCR) usingplasmid pEGFP-C2 (Clonetec) to amplify the complete sequence of GFP. Inthis PCR, the sequence of a sense primer was5′-CTCGAGGTGAGCAAGGGCGAGGAGCTG-3′ (SEQ ID NO: 1), and the sequence of anantisense primer was 5′-GGATCCTTACTTGTACAGCTCGTCC ATGCCGAG-3′ (SEQ IDNO: 2). The PCR product was cut with XhoI-BamHI, and subcloned into theXhoI-BamHI site of pET15b (Invitrogen, Carlsbad, Calif.) to produce pGFPexpressing a GFP fusion protein having no basic domain of HIV-1 Tat. Aclone having about 0.7 kb insert was selected by XhoI-BamHI restrictionenzyme analysis and sequenced.

pTat-GFP expressing the basic domain (amino acids 48-57) of HIV-1 Tatfused to GFP was produced in the following method. First, twooligonucleotides were produced and annealed into double-strandedoligonucleotides coding for 9 amino acids of the basic domain of HIV-1Tat. The sequences coding for 9 amino acids of the basic domain of HIV-1Tat were a top strand of 5′-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3′ (SEQ ID NO:3) and a bottom strand of 5′-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3′ (SEQ IDNO: 4). The double-stranded oligonucleotides were connected directly toa site of pGFP digested with NdeI-XhoI. Thus, Tat-GFP expression plasmidpTat-GFP which had been connected with 6-histidine open reading frame inframe, was produced (FIG. 1).

Furthermore, to produce pTat-GFP-Tat where the Tat basic domain wasfused to both terminal ends, a double-stranded oligonucleotide codingfor 9 amino acids of the Tat basic domain was annealed into pTat-GFP andinserted into the carboxyl-terminal end. To produce pGFP-Tat where theTat basic terminal end was fused to the carboxyl-terminal end, adouble-stranded oligonucleotide coding for 9 amino acid of the Tat basicdomain was annealed into pGFP and inserted into the carboxyl terminalend (FIG. 1).

The sequence of the oligonucleotide cloned into the plasmid was analyzedwith a fluorescence-based automated sequencer (Model 373A, AppliedBiosystems, Inc.).

EXAMPLE 2 Expression and Purification of Tat-GFP, GFP-Tat andTat-GFP-Tat Fusion Proteins (Native and Denatured States)

E. coli BL21 (Pharmacia) transformed with pGFP and pTat-GFP, pGFP-Tatand pTat-GFP-Tat, and the like, was selected, and then the colony wasinoculated to an LB medium containing 100 μg/ml ampicillin and culturedat 37° C. overnight. The cultured medium was 10-fold diluted in a freshLB medium and cultured with stirring at 250 rpm. When the bacterialconcentration (the optical density at a 600 nm wavelength) in thecultured medium reached 1.0, IPTG was added into the cultured medium toa final concentration of 0.5 mM and then the cells were cultured for 4hours.

To obtain the cultured cells were added to a denatured transductiondomain-GFP fusion protein, a binding buffer (5 mM imidazole, 0.5M NaCl,20 mM Tris-HCl, pH 7.9) containing 6M urea and a protease inhibitor (20mg/ml soybean trypsin inhibitor, 2 mg/ml aprotinin, 5 mg/ml leupeptin,100 mg/ml PMSF). The mixture was subjected to sonication, harvested andlysated. The resulting substance was placed in buffer A (6M urea, 20 mMHEPES, pH 8.0, 100 mM NaCl), subjected to sonication to disrupt thecell, and then subjected to two consecutive centrifugations (at 16,500rpm for 30 min, and then 40,000 rpm for 30 min, each at 4° C.) to removeinsoluble cell debris. The lysate was purified through a Ni⁺⁺⁺-IDAcolumn. The column was washed with a binding buffer containing no 6Murea, and then with a washing buffer (80 mM imidazole, 0.5M NaCl, 20 mMTris-HCl, pH 7.9). The protein was eluted with an elution buffer (1 mMimidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9), and then purified throughPD-10 column chromatography (Amersham) to remove salts contained in theprotein (FIG. 2). The purified GFP fusion protein was expressed andpurified into about 30 kDa size, the Tat-GFP and GFP-Tat fusion proteinswere expressed and purified to a size as large as the size (about 1 kDa)of the Tat transduction domain, and the Tat-GFP-Tat fusion protein hadthe Tat transduction domains fused to both sides thereof, and thusexpressed and purified to a molecular weight greater than the Tat-GFPand GFP-Tat fusion proteins by the size of one Tat transduction domain.

A native, transduction domain-GFP fusion protein was produced by thesame procedures without the above-mentioned denaturing agent. Eachfraction was isolated by SDS-PAGE and then quantified using a bovineserum albumin (BSA) standard by an optical density measurement method.The protein concentration was determined by Bradford protein assay(Biorad). The purified proteins were dissolved in PBS containing 20%glycerol, and stored at −80° C.

EXAMPLE 3 Culturing of Cells and Test of Intracellular Transduction ofFusion Proteins

HeLa cells were cultured in DMEM (Dulbecco's modified eagle's medium)containing 20 mM HEPES/NaOH (pH 7.4), 5 mM NaHCO3, 10% fetal bovineserum (FBS) and antibiotics (100 μg/ml streptomycin, 100 U/mlpenicillin) at 37° C.

In order to observe the intracellular transduction of a transductiondomain fusion protein where a HIV-1 Tat protein transduction domainconsisting of 9 amino acids (49-57) was bound to the amino-terminal endand/or the carboxyl-terminal end, the following test was performed.Namely, in order to observe the ability of transduction domain-GFP,GFP-transduction domain, transduction domain-GFP-transduction domainfusion proteins to be transduced into cells, the HeLa cells were grownon a 6-well plate for 4-6 hours, and replaced by a fresh DMEM culturemedium containing 10% FBS, and then GFP fusion proteins were added tothe cultured medium at various concentrations. After culturing at 37 □for 1 hour, the cells were sufficiently washed with phosphate bufferedsaline (PBS), and treated with trypsin-EDTA (Gibco BRL) for 10 minutes.After the cells were disrupted, the amount of the transductiondomain-GFP fusion protein transduced into the cell was measured byWestern blot analysis as described in the following example.

EXAMPLE 4 Western Blot Analysis

In order to analyze the efficiency of intracellular transduction of thefusion protein where one Tat transduction domain is fused to theamino-terminal end as well as the fusion protein where Tat transductiondomains are fused to both terminal ends, Western blot analysis wasperformed as follows. First, proteins were purified in a denaturedstate. Prepared animal HeLa cells were treated with 1M of the denaturedproteins, and after one hour, only the cells were collected andsubjected to Western blot analysis. The cells were lysated, and theresulting cell lysates were placed into a 6-well plate containing anelution buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 10% v/v glycerol). Each15 μg of the cell lysates was electrophoresed on SDS-12% polyacrylamidegel.

The protein separated by the electrophoresis was transferred to anitrocellulose membrane (Amersham, UK). The nitrocellulose membrane towhich the protein had been transferred was blocked with PBS containing10% dry milk. Then, the membrane was treated with a rabbit anti-GFPpolyclonal antibody (Clontech, USA, 1:1,000). Next, it was reacted witha Horseradish peroxidase-bound goat anti-rabbit IgG antibody (Sigma,1:10,000 dilution). The bound antibodies could be detected by enhancedchemiluminescence (ECL; Amersham). As shown in FIG. 3, the resultsshowed that the fusion protein having the Tat transduction domain onlyat the amino-terminal end and the fusion protein having the Tattransduction domain at the carboxyl-terminal end were transduced intothe cells in similar amounts. However, the protein where the Tattransduction domains had been fused to both terminal ends was transducedinto the cell in a more efficient manner than the fusion protein towhich one Tat transduction domain had been fused. Such results indicatethat whether the Tat transduction domain is fused to the amino-terminalend or fused to carboxyl terminal end of the fusion proteins, it showssimilar effects on the intracellular delivery of the target protein, butif the Tat transduction domain is fused to both terminal ends of thefusion protein, its effect on the intracellular delivery of the targetprotein will be greatly improved.

EXAMPLE 5 Analysis of Intracellular Transduction by Confocal Microscopy

In transducing fusion proteins into target cells, the ratio of the cellsinto which the fusion proteins are transduced is important. Furthermore,only if the fusion proteins transduced into the cells maintain theirinherent activity, this protein transduction technology can be applied.Thus, in this example, the ratio of cells into which the GFP fusionprotein is transduced, or whether the transduced fusion protein hasactivity or not, was analyzed. The fluorescent intensity of GFP in HeLacells treated with a denatured GFP fusion protein was observed with afluorescent microscope.

The cells were cultured to 50-70% confluency on a cover slip, andtreated with 1M of a GFP-fusion protein having transduction domainsbound to both terminal ends thereof, and then cultured for 15 minutes.The HeLa cells were washed two times with PBS, and treated with trypsin,and then immobilized with PBS containing formaldehyde at roomtemperature for 15 minutes. The cells were washed with PBS again andtreated with 2 g/ml of propidium iodide (PI) for 15 minutes to stainonly nuclei. The cells were washed with PBS again, and treated with amounting solution (phosphate buffered saline (PBS) containing 90%glycerol and 0.1% phenylenediamine), and covered with a cover glass.Then, whether the cells have fluorescence or not was observed with 488nm and 545 nm fluorescent filters using a confocal fluorescentmicroscope to analyze the distribution of the proteins in the cells(Eric et al., 1997). The results showed that the protein having the Tattransduction domain fused to the amino-terminal end, and the proteinhaving the Tat transduction domain fused to the carboxyl-terminal end,reached both cytoplasms and nuclei in substantially similar manners.Similarly to the results of the Western blot analysis, this suggeststhat whether the Tat transduction domain is fused to the amino-terminalend or fused to the carboxyl-terminal end of the proteins, it showssimilar effects on the intracellular delivery of the target protein. Onthe other hand, the fusion protein having the Tat transduction domainsfused to both terminal ends thereof showed a higher fluorescenceintensity than the proteins having the Tat transduction domain only atone terminal end. In addition, the fusion protein having the Tattransduction domains fused to both terminal ends was transduced intonuclei at larger amounts.

EXAMPLE 6 Analysis of Transduction into Cells by FACS (Flow Cytometry)Analysis

In order to observe the intracellular transduction of a transductiondomain fusion protein where HIV-1 Tat protein transduction domains eachconsisting of 9 amino acids (49-57) were bound to the amino terminal endand carboxyl terminal ends, the following test was performed (FIG. 5).Namely, in order to observe the ability for the transductiondomain-GFP-transduction domain fusion protein to be transduced intocells, HeLa cells were grown on a 6-well plate for 4-6 hours, andreplaced by a fresh DMEM medium containing 10% FBS, and then GFP fusionproteins were added to the cultured medium at various concentrations.After culturing at 37° C. for 30 minutes, the cells were sufficientlywashed with phosphate buffered saline (PBS), and treated withtrypsin-EDTA (Gibco BRL) for 10 minutes. The cells were collected,washed twice with PBS again, and immobilized with 4% paraformaldehydefor 1 hour. The intracellular fluorescence of the immobilized cells wasanalyzed by FACS analysis. According to the results of the FACSanalysis, curves as shown in FIG. 5 were obtained. Similarly to theresults as described above, it could be found that the proteins havingthe Tat transduction domain fused to one terminal end thereof wereintroduced into the cells at similar amounts whereas the fusion proteinhaving the Tat transduction domains fused to both terminal ends thereofwas introduced into increased amounts. Mean fluorescence intensity was23.86 for the transduction domain-GFP fusion protein, 17.21 for theGFP-transduction domain fusion protein, and 204.52 for the transductiondomain-GFP-transduction domain fusion protein, indicating that thetransduction domain-GFP-transduction domain shows significantlyincreased fluorescence intensity. Then, in order to analyze themechanism of intracellular transduction of the Tat-GFP-Tat fusionprotein on the basis of its amount, the Tat-GFP-Tat fusion protein wasadded to the cells at various concentrations, and the mixture wascultured for 30 minutes and then subjected to FACS analysis (FIG. 6). Asa result, it could be found that the fusion protein having the Tattransduction domains fused to both terminal ends thereof showed anincrease in mean fluorescence intensity with an increase in itsconcentration.

EXAMPLE 7 FACS (Flow Cytometry) Analysis and Western Blot Analysis forComparison Between Intracellular Transduction of Native Fusion Proteinand Denatured Fusion Protein

A transduction domain-GFP fusion protein which was purified in a nativestate is significantly limited in intracellular transduction as comparedto a transduction domain-GFP fusion protein which was purified in adenatured state. When purified in a denatured state, the GFP fusionprotein having the transduction domains fused to both terminal endsthereof shows a higher effect than the GFP fusion protein having thetransduction domain fused to one terminal end thereof. For this reason,in order to examine the effect of intracellular transduction of thetransduction domain-GFP-transduction domain fusion protein purified in anative state, the following tests were performed. First, a proteinpurified in a denatured state, and a protein purified in a native state,were analyzed by Western blot analysis. The results showed that theprotein purified in a native state was transduced into cells at the samelevel as the GFP fusion protein having one transduction domain (FIG. 7).Also, in order to examine the effect of intracellular transduction ofthe transduction domain-GFP-transduction domain fusion protein purifiedin a native state, the following test was performed. Each of the proteinpurified in a native state and the protein purified in a denatured statewas added to cells at various concentrations and then the cells werecultured for 1 hour. As shown in FIG. 8, when the transductiondomain-GFP-transduction domain fusion protein purified in a denaturedstate was added to cells at 1M, the mean fluorescence intensity of thecells was 117, but when the transduction domain-GFP-transduction domainfusion protein purified in a native state was added to cells, the meanfluorescence intensity of the cells was 54. This suggests that theeffect of intracellular transduction of the transductiondomain-GFP-transduction domain fusion protein is inferior to that of thetransduction domain-GFP-transduction domain fusion protein purified in adenatured state, but equal to that of the transduction domain-GFP fusionprotein purified in a denatured state as shown in FIG. 7. As a result,it can be concluded that the fusion protein, which has the Tattransduction domains at both terminal ends thereof and was purified in adenatured state, can be transduced into cells in a more effective mannerthan the fusion protein having the transduction domain at one terminalend, and the transduction domain-GFP-transduction domain fusion proteinpurified in a native state can also be transduced into cells.

EXAMPLE 8 Production of Vectors for Expression of Recombinant SOD FusionProteins

In order to develop a technology of delivering functional proteins orpeptides into cells, fusion protein expression vectors allowing thetarget proteins to be delivered into cells were produced. To facilitatethe analysis of the ability of the transduction domain to deliver theproteins into cells, a human Cu/Zn-superoxide dismutase (hereinafter,abbreviated to SOD) protein was selected.

To overexpress recombinant fusion proteins, Tat-SOD, SOD-Tat andTat-SOD-Tat expression vectors each containing SOD, HIV-1 Tattransduction domain (amino acids 49-57) and cDNA for 6 histidines inorder were produced (FIG. 9). To overexpress protein SOD as a controlfor fusion proteins, a pET-SOD expression vector containing the samedomains as the above expression vectors except for the Tat transductiondomain was produced.

Two oligonucleotides corresponding to a Tat basic domain and having atop strand of 5′-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3′ (SEQ ID NO: 3) and abottom strand of 5′-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3′ (SEQ ID NO: 4)were cut with NdeI-XhoI restriction enzyme, and ligated into a pET-15bvector, and then two oligonucleotides were synthesized on the basis ofthe sequence of a human SOD cDNA. The forward primer has an XhoIrestriction site, and the reverse primer has a BamHI restriction site.After performing polymerase chain reaction (PCR), the PCR product wasisolated, and transformed with a TA cloning vector, thereby producing aplasmid. The human SOD cDNA transformed with the TA vector was cut withXhoI and BamHI and inserted into pET-15b and pET-15b-Tat expressionvectors.

Furthermore, to produce a pTat-SOD-Tat expression vector having Tatbasic domains fused to both terminal ends thereof, double strandedoligonucleotide coding for 9 amino acids of the Tat basic domain wasannealed into pTat-SOD. To produce a pSOD-Tat expression vector having aTat basic domain fused to the carboxyl terminal end, double-strandedoligonucleotide coding for 9 amino acids of the Tat basic domain wasannealed into pSOD (FIG. 9).

EXAMPLE 9 Expression and Purification of Recombinant SOD Fusion Proteins

E. coli BL21 (DE3) cells (pSOD, pTat-SOD, pSOD-Tat, and pTat-SOD-Tat)which contain human Cu/Zn-superoxide dismutase cDNA and were produced bythe present inventors, were placed in an LB medium containingampicillin, and then the cells were cultured with stirring at 37° C. and200 rpm. When the bacterial concentration (the optical density at a 600nm wavelength) in the cultured medium reached 0.5-1.0, IPTG was added tothe medium to a final concentration of 0.5 mM, and then the cells werecultured for 3-4 hours. The cultured cells were centrifuged, collected,added with 5 ml of a binding buffer (5 mM imidazole, 0.5M NaCl, 20 mMTris-HCl, pH 7.9) containing 6M urea, and then disrupted by sonication.Immediately after centrifugation, the supernatant was loaded on aNi-nitrilotriacetic acid Sepharose superflow column, and washed with a10-fold volume of binding buffer and a 6-fold volume of washing buffer(60 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9), and then thefusion protein was eluted with elution buffer (1M imidazole, 0.5M NaCl,20 mM Tris-HCl, pH 7.9). Next, the fractions containing the fusionprotein were collected and purified by PD-10 column chromatography toremove salts contained in the fractions. Since the fusion proteincontained 6 histidines, it was purified to a purity of more than 90% bymetal ion-chelate affinity chromatography, single stage (FIG. 10).

The concentration of protein in the fractions was measured by a Bradfordmethod using a fetal serum albumin standard.

EXAMPLE 10 Culturing of HeLa Cells and Intracellular Transduction ofRecombinant Fusion Proteins

HeLa cells were cultured in DMEM (Dulbecco's modified eagle's medium)containing 20 mM HEPES/NaOH (pH 7.4), 5 mM NaHCO₃, 10% fetal bovineserum (FBS) and antibiotics (100 μg/ml streptomycin, 100 U/mlpenicillin) with supply of 95% air and 5% CO₂ at 37° C.

In order to the efficiency of intracellular transduction of transductiondomain fusion proteins where a HIV-1 Tat protein transduction domainconsisting of 9 amino acids (49-57) were bound to the amino-terminal endand/or the carboxyl-terminal end, the following test was performed.Namely, in order to observe the ability of Tat-SOD, SOD-Tat, andTat-SOD-Tat fusion proteins to be transduced into cells, HeLa cells weregrown on a 6-well plate for 4-6 hours, and then replaced by a fresh DMEMculture medium containing 10% FBS, and the cultured medium was treatedwith 2M SDS fusion proteins. After culturing the cells at 37° C. for 1hour, the cells were sufficiently washed with phosphate buffered saline(PBS), and treated with trypsin-EDTA (Gibco BRL) for 10 minutes. Afterthe cells were disrupted, the amount and activity of the SOD into thecells were measured by SOD activity analysis and Western blot analysis.

EXAMPLE 11 Western Blot Analysis

In order to analyze the efficiency of intracellular transduction of aprotein where one Tat transduction domain is fused to the amino-terminalend as well as a protein where Tat transduction domains are fused toboth terminal ends, Western blot analysis was performed as follows.First, proteins were purified in a denatured state. Prepared HeLa cellswere treated with 2M of the denatured fusion proteins, and after onehour, only the cells were collected and subjected to Western blotanalysis. The cells were lysated, and the resulting cell lysates wereplaced into a 6-well plate containing a lytic buffer (125 mM Tris-HCl,pH 6.8, 2% SDS, 10% v/v glycerol). Each 15 μg of the cell lysates waselectrophoresed on SDS-12% polyacrylamide gel.

The protein separated by the electrophoresis was transferred to anitrocellulose membrane (Amersham, UK). The nitrocellulose membrane towhich the protein had been transferred was blocked with PBS containing5% non-dry milk. Then, the membrane was treated with a goat anti-SODpolyclonal antibody (Santacruz, USA, 1:1,000). Next, it was reacted witha Horseradish peroxidase-bound mouse anti-rabbit IgG antibody (Sigma,1:10,000 dilution). The bound antibodies could be detected by enhancedchemiluminescence (ECL; Amersham). As shown in FIG. 11 a, the resultsshowed that the fusion protein having the Tat transduction domain onlyat the amino-terminal end and the fusion protein having the Tattransduction domain at the carboxyl-terminal end, were transduced intothe cells in similar manners. However, the protein having the Tattransduction domains fused to both terminal ends thereof was transducedinto the cells in a more effective manner than the fusion protein towhich one Tat transduction domain is fused. Such results indicate thatwhether the Tat transduction domain is fused to the amino terminal endor fused to carboxyl terminal end of the fusion protein, it showssimilar effects on the intracellular delivery of the target protein, butif the Tat transduction domain is fused to both terminal ends of thefusion protein, its effect on the intracellular delivery will be greatlyimproved (FIG. 11 a).

EXAMPLE 12 Measurement of Activity of SOD Enzyme

In this example, the activity of SOD was measured by observing thereduction of ferricytochrome c by xanthine/xanthine oxidase with aspectrophotometer according to the method of McCord and Fridovich(McCord, J M and Fridovich, I., J. Biol. Chem. 244, 6049-6055 (1969)).

The standard analysis method was performed in 50 mM of a phosphatebuffered solution (pH 7.8) containing 2 ml of 0.1 mM EDTA at 25° C. Thereaction mixture contained 10 μM ferricytochrome c, 50 μM xanthine and asufficient amount of xanthine oxidase, and the reduction offerricytochrome c was measured at 550 nm. The amount of superoxidedismutase at which cytochrome c is reduced by 50% was defined as 1 unit.

In order to make it possible to apply the fusion proteins for proteintherapy, the fusion proteins transduced into cells must maintain theirinherent activity. Thus, it is a very important problem that the fusionproteins transduced into cells has biological activity. FIG. 11 b showsa change in enzymatic activity of SOD when 2M of fusion proteins wereadministered to a HeLa cell medium for 1 hour. The activity of SOD incells to which the fusion proteins had not been administered was2.46±0.39 U/mg protein, and the activity of SOD in cells to which theTat-SOD and SOD-Tat fusion proteins was 12.68±1.44 U/mg protein whichindicates a significant increase in the SOD activity. When theTat-SOD-Tat fusion protein was administered to cells, the SOD activitywas 23.75±2.35 U/mg protein which indicates a two times increase in theSOD activity as compared to that of the Tat-SOD and SOD-Tat fusionproteins (FIG. 11 b). As a result, it could be found that theintracellular transduction and activity of the Tat-SOD-Tat fusionprotein was at least two times higher than the Tat-SOD and SOD-Tatfusion proteins (FIG. 11).

EXAMPLE 13 Measurement of Ability of Arginine 9-GFP-arginine 9 FusionProtein to be Transduced into Cells

According to a similar method to Examples 1-3, an arginine-GFP-argininefusion protein where the transduction domains each consisting of 9arginine residues had been covalently bonded to the amino- andcarboxyl-terminal ends of the green fluorescent protein (GFP) wasproduced.

In order to compare the efficiency of intracellular delivery of theTat-GFP-Tat fusion protein and the arginine 9-GFP-arginine 9 (R9-GFP-R9)fusion protein, each of the fusion proteins were added to a 24-wellplate containing HeLa cells, at 50 nM, 100 nM and 200 nM. After onehour, the cells were treated with trypsin, washed several times withPBS, and measured for fluorescence with a fluorometer. The results areshown in FIG. 12.

EXAMPLE 14 Measurement of Ability of Lysine 9-GFP-lysine 9 FusionProtein to be Transduced into Cells

According to a similar method to Examples 1-3, anoligolysine-GFP-oligolysine fusion protein where the transductiondomains each consisting of 9 lysine residues are covalently bonded tothe amino and carboxyl terminal ends of the green fluorescent protein(GFP) was produced.

In order to compare the efficiency of intracellular delivery of theTat-GFP fusion protein, the GFP-Tat fusion protein, the arginine 9-GFPfusion protein and the lysine 9-GFP-lysine 9 (K9-GFP-K9) fusion protein,200 nM of each of the fusion proteins was added to a 24-well platecontaining HeLa cells. After one hour, the cells were treated withtrypsin, washed several times with PBS, and measured for fluorescencewith a fluorescent microscope. The results are shown in FIG. 13.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides the fusion proteinwhich delivers the functional proteins into cells at increasedefficiency and has increased activity in the cells, as compared to theexisting fusion proteins having the basic transduction domain at oneterminal end.

Furthermore, the present invention provides pharmaceutical compositionand cosmetics, etc., which contain the inventive fusion protein as anactive ingredient.

Moreover, the present invention proposes that the transductiondomain-SOD-transduction domain, which delivers the humanCu/Zn-superoxide dismutase directly into cells at a protein level, canbe efficiently applied to protein therapy, since it shows increased SODactivity when delivered into the cells.

The reactive oxygen species cause damage to biopolymers, and asreported, they have a deep connection with about 10 kinds of diseases.Thus, according to the present invention, the transductiondomain-SOD-transduction domain fusion protein of the present inventioncan be effectively used in protein therapy where the SOD playing a mainrole in removing such reactive oxygen species is delivered into cells totreat diseases.

According to the present invention, the SOD that is a kind ofantioxidant enzymes can be delivered into cells to remove the reactiveoxygen species harmful to the human body. Thus, the present inventioncan be used in a wide range of industrial fields, including the cosmeticand health food industries, in addition to treating various diseases.

1. A transduction domain-target protein-transduction domain fusionprotein having the ability to be transduced into a cell, wherein thetarget protein is a Cu/Zn-superoxide dismutase and the transductiondomains comprise HIV tat residues 49-57 and are covalently bonded toeach of the amino- and carboxyl-terminal ends of the target protein. 2.A cosmetic composition comprising the transduction domain-targetprotein-transduction domain fusion protein of claim 1 as an activeingredient.
 3. The cosmetic composition of claim 2, which is in the formof toilet water, gel, water-soluble liquid, oil-in-water (O/W), orwater-in-oil (W/O).
 4. A pharmaceutical composition comprising: thetransduction domain-target protein-transduction domain fusion protein ofclaim 1 as an active ingredient, and a pharmaceutically acceptablecarrier.